Novel Coated Membranes and Other Articles

ABSTRACT

The present invention provides porous media or membranes having a surface coating that includes a cross-linked terpolymer which has a superior combination of properties, including heat stable biomolecule resistant adsorptive properties, resistance to strong alkaline solutions, and low levels of extractable matter. In some preferred embodiments, the porous media is a porous membrane.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority benefit of U.S. Provisional ApplicationSer. No. 60/287,172 filed Apr. 27, 2001, the contents of which areincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to the field of porous media having a bulkmatrix of a first material and a surface coating of a second material.The surface coating comprises a cross-linked terpolymer which has asuperior combination of properties, including heat stable biomoleculeresistant adsorptive properties, resistance to strong alkalinesolutions, and low levels of extractable matter. In some preferredembodiments, the porous media is a porous membrane.

BACKGROUND OF THE INVENTION

Porous media are useful in many separation and adsorption technologies,such as chromatography. One particular type of porous media, porousmembranes, are used for a variety of applications. Porous membranes havea first porous surface, a second porous surface, and a continuous porousstructure that extends throughout the membrane from the first to thesecond surface. The continuous porous structure includes the bulkmaterial matrix and the network of pores. The interface separating thebulk matrix from the pore volume (i.e., the surface of the interior porenetwork) is known as the interstitial surface.

Porous membranes can be classified as “microporous” membranes or“ultrafiltration” membranes on the basis of the size of the pores of themembrane. Generally, the range of pore sizes for microporous membranesis considered to be from approximately 0.05 micron to approximately 10.0microns, whereas the range of pore sizes for ultrafiltration membranesis considered to be from approximately 0.002 micron to about 0.05micron. These pore sizes refer to pore diameter for circular orapproximately circular pores, or to a characteristic dimension fornon-circular pores.

The pore size of a membrane can be denominated by the size of thesmallest species (particle or molecule) that cannot pass through themembrane above a specified fractional passage. A common rating is below10% passage, which corresponds to a 90% cutoff or retention. Othermethods are known to those skilled in the art, including image analysisof scanning electron microscopy to obtain pore size distributioncharacteristics. Microporous membranes are typically used to removeparticulates from liquids and gases. An important application ofmicroporous membranes is in sterile filtration of pharmaceuticalsolutions to remove any bacteria that may be present in the solution.Microporous membranes are also used as sterile gas vents, which allowgas flow but prevent airborne bacteria from passing through the filter.Ultrafiltration membranes are generally used in applications whereretention of smaller species is desired. For example, ultrafiltrationmembranes are used in the biotechnology industry to concentrateproteins, and in diafiltration applications to remove salts and lowmolecular weight species from protein solutions. Both ultrafiltrationand microporous membranes can be fabricated in several forms, includingsheets, tubes, and hollow fibers.

Porous membranes are made from a variety of materials, polymers beingthe most common. Many commercial membranes are made from engineeringplastics, such as polyethersulfone, polysulfone, polyvinylidenefluoride, polyethylene, polytetrafluoroethylene, polypropylene and soforth, to take advantage of their robust thermal, mechanical, andchemical-resistance properties. Unfortunately, these materials arehydrophobic and have a high propensity to adsorb biomolecules.

In general, a hydrophilic membrane is preferred for filtration ofaqueous solutions. This is because with hydrophobic membranes, contactwith a low surface tension prewetting liquid followed by water exchangeis required to start permeation. This not only introduces added materialcost to the process, but any wetting liquid must be exhaustivelyflushed, which greatly increases process time and cost.

In addition to permeability and retentive properties, porous membraneshave other requirements that are dictated by the nature of theapplication. For example, they must have sufficient mechanical strengthto withstand operating pressures and temperatures. In applications wherecleanliness is a major requirement, as in the pharmaceutical ormicroelectronics wafer-making industry, the amount of material thatextracts from the membrane in use must be very small. In applicationswhere the membrane comes in contact with biomolecules, the membranesurface must be resistant to biomolecule adsorption. A biomoleculeresistant surface is a surface that adsorbs or binds minimal amounts ofbiomolecules, such as proteins and nucleic acids; specifically, asurface that adsorbs less than about 30 micrograms of IgG per squarecentimeter of membrane area as measured by the IgG binding testdescribed herein. It is greatly preferred that a membrane surface bemaximally biomolecule resistant, to reduce permeation loss from foulingby surface adsorption or pore blockage, and to prevent product loss byirreversible adsorption or associated biomolecule denaturization.

In many applications, porous membranes comes in contact with highlyalkaline solutions in cleaning or sanitation operations. Thus, themembrane must have sufficient chemical resistance to withstand suchcontact without loss of filtration properties or desirable surfaceproperties.

To impart the aforementioned properties to porous media or membranes,manufacturers typically modify the membrane surface (i.e., the first andsecond surfaces and the interstitial surface) of the bulk matrixmaterial making up the porous media or membrane to make the surfacehydrophilic and biomolecule resistant. This is done by a variety ofprocedures that coat, attach to, or otherwise cover the surface of thebulk matrix material with a hydrophilic polymer or group. While suchmodification can solve some problems related to the hydrophobic or highbiomolecule binding of the bulk matrix material surface, it also can addother problems. For example, such modifications increase the amount ofmaterial able to be extracted from the membrane during use, and themodification material can have low tolerance to exposure to alkalinesolutions. In addition, in many applications membranes are heated,either by wet heat as in autoclaving or steam sanitization, or by dryheat, as in a drying step. It has been observed that such heating willreduce hydrophilicity and decrease biomolecule resistance of somemodified surfaces.

Some membranes of the prior art are made by modifying the surface ofpreformed porous membranes with cross-linked hydroxyacrylates, where thecrosslinking monomer is a difunctional acrylate (“Case A membranes”).Such membranes have excellent resistance to biomolecule adsorption,excellent heat stability of the biomolecule resistance, and acceptableflow loss (i.e., the reduction in flow or permeability compared to theunmodified membrane). However, while these membranes have acceptablecleanability characteristics (i.e., the ability to clean the membrane bywashing such that residual extractable matter (“extractables”) islowered to an acceptable level, it was found that to lower extractablesto below a certain level, about 2 microgram per square centimeter usingthe TOC test (described herein in the “Methods” section), a veryextensive extraction regime was needed. In addition, these membraneswere sensitive to strong alkaline solutions.

Much of the prior art describes the use of hydroxyl containing monomers,usually carbonyl ester containing acrylate polymers, to produce membranesurface modifications having hydrophilic character and high resistanceto protein binding. However, it is known that polymers from suchmonomers are not resistant to strong alkaline solutions. For example, asolution of 1.0 normal sodium hydroxide will hydrolyze the carbonylcontaining acrylate polymers to acrylic acid containing polymers. Suchacrylic acid containing polymers are ionically charged under certain pHconditions, and will attract and bind oppositely charged proteins orbiomolecules, thus increasing sorption and membrane fouling. Inaddition, acrylic acid containing polymers swell in water such that theyconstrict pore passages, thus reducing membrane permeability andproductivity. Moreover, polymers from hydroxyl containing monomers, suchas hydroxy acrylates, further react in strong alkaline solutions anddegrade into soluble low molecular weight fragments, which dissolve awayand expose the underlying porous media or membrane.

Various methods of modifying porous membranes are known in the art. Forexample, U.S. Pat. No. 4,618,533 discloses and claims a composite porousthermoplastic membrane which comprises a porous membrane substratehaving an average pore size between about 0.001 and 10 microns formed ofa first polymer, the substrate being directly coated on its entiresurface with a cross-linked second polymer formed from a monomerpolymerized in situ with a free radical initiator on the substrate,where the composite porous membrane has essentially the same porousconfiguration as the membrane substrate.

U.S. Pat. No. 4,944,879 discloses a composite porous membrane havingdesired bulk properties on which is coated a cross-linked polymer havingdesired surface priorities. The cross-linked surface polymer is producedfrom a crosslinkable monomer or polymer by energy from an electron beamin the absence of a chemical polymerization initiator.

Similar modified porous media are disclosed in U.S. Pat. Nos. 4,906,374,4,968,533, and 5,019,260, in which hydroxyl containing polymericmaterial is derived from monomers having hydroxyl groups and moietiescharacterized by having one polymerizable unit of unsaturation, such ashydroxy or hydroxy-forming substituted acrylates or methacrylate esters.Polymers from such monomers are known to lack resistance to degradationby strong alkaline solutions.

Chapman et al (J. Am. Chem. Soc. 2000, 122, 8303-8304) describe the useof self assembled monolayers (SAM) to screen functional groups forprotein resistance. They report several functional groups to be proteinresistant, including N-substituted amide functionalities.

U.S. Pat. Nos. 4,695,592 and 4,678,813 describe a process and productfor a hydrophilized porous polyolefin membrane with a crosslinkedpolymer, which is composed of 50% or more of diacetone acrylamidemonomer.

Iwata et al (J. Membrane Sci. 1991 55 119-130) report acrylamidecoatings of membranes that have temperature responsive properties (TRP),specifically polyacrylamides, and particularlypoly(N-isopropylacrylamide (polyIPAA)). Iwata report the graftpolymerization of homopolymers of polyIPAA and copolymers withacrylamide to a first surface of a PVDF membrane. However, they do notcross-link the polymers, as that would impede the polymer TRP.

U.S. Pat. No. 5,929,214 to Peters et al, describes porous monolithsfunctionalized and/or grafted with TRP polymers, includingnon-crosslinked copolymers of polyIPAA. These membranes are designed toadsorb biomolecules, and the Peters et al. patent does not teach theproduction of protein or biomolecule resistant surfaces.

It can be seen that practitioners attempting to develop optimizedmembranes for sterile filtration and other applications in thepharmaceutical and biotechnology industries have to overcome significantproblems. Facing stringent cost, performance and safety requirements, apractitioner has to use materials and develop manufacturing methods thatproduce membranes with not only optimized flow and retentioncharacteristics, but be economical to produce, meet cleanlinesscriteria, be stable to the various chemical environments which arecommonly encountered, and be very resistant to biomolecule adsorption.Thus, it would be desirable to have a membrane modification that resultsin a hydrophilic, biomolecule resistant surface that is heat stable,which is resistant to degradation by alkaline solutions, and which hasvery low levels of material capable of being extracted therefrom. Thisinvention is directed to these, as well as other, important ends.

SUMMARY OF THE INVENTION

This invention is directed to polymeric porous media, preferably porousmembranes, that have been modified by forming in situ on the surfacethereof a cross-linked polymeric terpolymer coating. In some preferredembodiments, the coated porous media or membranes have substantially thesame porous character as the unmodified porous media or membrane, andalso have biomolecule resistant sorptive properties, including heatresistant biomolecule resistance, chemical resistance to strong alkalinesolutions, and very low levels of extractable matter. In some morepreferred embodiments, the modified porous media or membrane ishydrophilic, and does not substantially change pore size as a functionof temperature.

Thus, in some preferred embodiments, the present invention providesclean porous membranes comprising a porous substrate and a separatelyformed heat stable biomolecule resistant surface. In further preferredembodiments, the present invention provides clean porous membranescomprising a porous substrate and a separately formed caustic resistant,heat stable biomolecule resistant surface.

In some preferred embodiments, the porous support and the polymercoating are formed from different materials.

Preferably, the porous substrate is a membrane, more preferably amicroporous membrane.

In further preferred embodiments, the invention provides clean, causticresistant, porous membranes comprising a microporous membrane substratewhich is preferably formed from one or more of the group consisting ofaromatic sulfone polymers, polytetrafluoroethylene, perfluorinatedthermoplastic polymers, polyolefin polymers, ultrahigh molecular weightpolyethylene, and polyvinylidene difluoride, and a heat stablebiomolecule resistant surface that is a separately formed surfacecoating which comprises a crosslinked terpolymer, said terpolymercomprising at least two monofunctional monomers selected from the groupconsisting of acrylamides, methacrylamides, and N-vinyl pyrrolidones,and at least one polyfunctional monomer selected from the groupconsisting of polyfunctional acrylamides, polyfunctionalmethacrylamides, and diacroylpiperazines.

In some more preferred embodiments, the invention provides clean,caustic resistant, porous membranes comprising a polyvinylidenedifluoride microporous membrane substrate and a heat stable biomoleculeresistant surface, wherein said heat stable biomolecule resistantsurface is a separately formed surface coating which comprises acrosslinked terpolymer, said crosslinked terpolymer being a copolymerformed from either:

-   -   (a) methylene-bis-acrylamide, dimethylacrylamide, and diacetone        acrylamide; or    -   (b) methylene-bis-acrylamide, vinylpyrrolidone, and either of        dimethylacrylamide or diacetone acrylamide.

Also provided in accordance with some preferred embodiments of thepresent invention are methods for the preparation of a clean, causticresistant porous membrane, said membrane comprising a porous membranesubstrate and a heat stable biomolecule resistant surface coating, saidmethod comprising the steps of:

-   -   a. providing a porous membrane substrate;    -   b. optionally washing said porous membrane substrate with a        wetting, liquid to wet the surfaces thereof;    -   c. optionally washing said wet porous membrane substrate with a        second wetting liquid to replace said first wetting liquid,        leaving said porous membrane substrate wetted with said second        liquid;    -   d. contacting the surfaces of said porous membrane substrate        with a reactant solution containing:        -   (1) at least two monofunctional monomers selected from the            group consisting of acrylamides, methacrylamides, and            N-vinyl pyrrolidones; and        -   (2) at least one polyfunctional monomer selected from the            group consisting of polyfunctional acrylamides,            polyfunctional methacrylamides and diacroylpiperazines;        -   said solution optionally further comprising one or more            polymerization initiators;    -   e. polymerizing said monomers to form said heat stable        biomolecule resistant surface; and    -   f. washing said membrane.

Preferably, the sizes of the pores of the porous substrate prior toperforming steps (a) through (e) are not significantly different fromthe sizes of said pores after performing steps (a) through (e). In somepreferred embodiments, the porous membrane substrate is a microporousmembrane.

In some preferred embodiments of the methods and membranes of theinvention where the porous substrate is a microporous membrane, themicroporous membrane is formed from one or more of the group consistingof aromatic sulfone polymers, polytetrafluoroethylene, perfluorinatedthermoplastic polymers, polyolefin polymers, ultrahigh molecular weightpolyethylene, and polyvinylidene difluoride, with polyvinylidenedifluoride being more preferred.

In some more preferred embodiments of the methods and membranes of theinvention, the crosslinked terpolymer comprises at least onemonofunctional monomer that is an acrylamide, wherein the acrylamidenitrogen of said acrylamide is substituted with at least one gem dialkylsubstituted carbon.

In some particularly preferred embodiments of the methods and membranesof the invention, the crosslinked terpolymer is a copolymer formed fromdimethylacrylamide, diacetone acrylamide, and methylene-bis-acrylamide.In other particularly preferred embodiments, the crosslinked terpolymeris a copolymer formed from methylene-bis-acrylamide, N-vinylpyrrolidone, and either of dimethylacrylamide or diacetone acrylamide.

In some preferred embodiments of the membranes of the invention, theheat stable biomolecule resistant surface of the membranes is aseparately formed surface coating comprising a crosslinked terpolymer;the crosslinked terpolymer comprising:

at least one polyfunctional monomer selected from the group consistingof polyfunctional acrylamide monomers, polyfunctional methacrylamidemonomers, and diacroylpiperazines; and

at least two different monofunctional monomers selected from the groupof N-vinyl pyrrolidone monomers and monomers having the general formula:

alternatively, described as H₂C═C(R¹)C(═O)N(R²) (R³)

wherein:

R¹ is —H or CH₃,

R² is H or C₁-C₆, preferably C₁-C₃ alkyl, either linear or branched,

R³ is H or C₁-C₆, preferably C₁-C₃ alkyl, either linear or branched, orC(CH₃)₂CH₂C(═O)CH₃, or (P═O)((NCH₃)₂)₂, or C=ON(CH₃)₂, or CH₂—O—R⁴,where R⁴ is C₁-C₅ alkyl, either linear or branched, or (CH₂—CH₂—O)n-R⁵,where R⁵ is —H or —CH₃, and n=2 or 3; provided that R² and R³ are notsimultaneously H.

In some preferred embodiments of the methods and membranes of theinvention, the crosslinked terpolymer of the membranes of the inventionfurther comprises a supplemental property modifying monomer, which ispreferably present in an amount that is less than either of themonofunctional monomers.

In some more preferred embodiments of the methods and membranes of theinvention, the supplemental property modifying monomer is selected fromthe group consisting of positively or negatively charged ion containingmonomers, monomers with affinity groups, or monomers with significanthydropohobic character. In further embodiments, the supplementalproperty modifying monomer is selected from the group consisting of(3-(methacryloylamino)propyl)trimethylammonium chloride,(3-acrylamidopropyl)trimethylammonium chloride,2-acrylamido-2-methyl-1-propanesulfonic acid andaminopropylmethacrylamide.

In some preferred embodiments of the methods and membranes of theinvention, two of the monofunctional monomers of the terpolymer arepresent in the weight ratio of about 1 to 5, with about 1 to 2 beingmore preferred.

In further preferred embodiments of the methods and membranes of theinvention, the total amount of monofunctional monomers present is fromabout 0.5% to about 20%, with from about 2% to about 10% being morepreferred, and from about 4% to about 8% being even more preferred.

In still further preferred embodiments of the methods and membranes ofthe invention, the ratio of the total amount of monofunctionalcomonomers to polyfunctional crosslinker monomer is about 1 to about 10,with about 2 to about 6 being more preferred.

In some preferred embodiments of the membranes of the invention, theheat stable biomolecule resistant surface is hydrophilic.

In some more preferred embodiments of the methods and membranes of theinvention, the membranes of the invention have a biomolecule binding ofless than about 30 microgram per square centimeter measured by the IgGbinding test.

In further preferred embodiments, the membranes of the invention haveTOC extractables of less than about 2.0 micrograms of extractable matterper square centimeter of membrane as measured by the NVR Extractablestest. More preferably, the membranes of the invention have less thanabout 1.5, more preferably less than about 1.4, more preferably lessthan about 1.3, more preferably less than about 1.2, more preferablyless than about 1.1, and even more preferably less than about 1.0micrograms of extractable matter per square centimeter of membrane asmeasured by the NVR Extractables test.

In further more preferred embodiments of the methods and membranes ofthe invention, the membranes of the invention have caustic resistance ofless than about 1.5, preferably less than about 1.3, more preferablyless than about 1.2, and even more preferably less than about 1.0 asmeasured by the Flow Time Measurement test.

In further preferred embodiments, the present invention also providesmethods for removing cells from a solution comprising the steps ofproviding a solution comprising having undesired cells; and filteringsaid solution through a membrane of the invention.

In still further preferred embodiments, the present invention alsoprovides methods for sterilizing a solution comprising the steps ofproviding a non-sterile solution and filtering said solution through amembrane of the invention.

In further embodiments, the present invention provides membranes havinga surface coating comprising at least onehydroxymethyldiacetoneacrylamide (HIMDAA) monomer of formula:

wherein R₁ and R₂ are each independently H or CH₂OH, preferably whereinR₁ and R₂ are each CH₂OH.

Also provided are methods for preparing a coated polymer membranecomprising the steps of:

-   -   a. providing a porous membrane substrate;    -   b. optionally washing said porous membrane substrate with a        wetting liquid to wet the surfaces thereof;    -   c. optionally washing said wet porous membrane substrate with a        second wetting liquid to replace said first wetting liquid,        leaving said porous membrane substrate wetted with said second        liquid;    -   d. contacting the surfaces of said porous membrane substrate        with a solution containing:

one or more monofunctional monomers, and

-   -   -   at least one monomer hydroxymethyldiacetoneacrylamide            (HMDAA) monomer of formula:

-   -   -   wherein R₁ and R₂ are each independently H or CH₂OH,            preferably wherein R₁ and R₂ are each CH₂OH; and

    -   e. polymerizing said monomers to form said coated membrane.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the levels of extractable material as total organic carbonfor membranes of the prior art, and of the present invention, afterdifferent soak times.

FIG. 2 illustrates the effect of heat on the resistance to biomoleculeadsorption of membranes of the present invention.

FIG. 3 shows the protein binding of membranes having surface coatingswhere HMDAA has been substituted in stepwise fashion for DAA.

FIG. 4 shows the protein binding of membranes having surface coatingswhere HMDAA has been substituted in stepwise fashion for MBAm.

DETAILED DESCRIPTION

The present invention provides porous membranes, preferably poroushydrophilic membranes, which have a superior combination of desirableproperties, including resistance to biomolecule adsorption (sometimesreferred to as “low affinity or sorbability for biomolecules” or“biomolecule resistance”), more preferably heat stable resistance tobiomolecule adsorption, chemical resistance to strong alkaline solutionssuch as are used to clean and/or sanitize porous membranes, and havingvery low levels of extractable matter than prior membranes. Inaccordance with the present invention, it has been discovered thatpolymeric porous media, preferably porous membranes useful forfiltration or as a diagnostic media, can be modified by forming in situa cross-linked terpolymer on the surface thereof to obtain a coatedmembrane having the aforementioned properties.

Thus, in some preferred embodiments, the present invention providespolymeric porous media, preferably a porous membrane, which has acrosslinked terpolymer surface. Preferably, the crosslinked terpolymersurface is formed in situ on the surface of the porous media ormembrane. Preferably, the modified porous media or membrane does notsubstantially change pore size as a function of temperature.

In preferred embodiments, the present invention provides porousmembranes having a heat stable biomolecule resistant surface. As usedherein, the term “biomolecule” is intended to mean any organic moleculethat is part of a living organism, or analogs thereof. Thus,biomolecules include polymers of amino acids, for example peptides andproteins (including antibodies and enzymes), and polymers of nucleotidessuch as DNA and RNA molecules, and DNA and RNA probes. Also includedwithin the definition of biomolecules are carbohydrates and lipids. Itis intended that synthetically produced analogs of each of the foregoingbe included in the definition of the term “biomolecule”.

As used herein, the terms “biomolecule resistant” or “biomoleculeresistance” as applied to membranes or membrane Surfaces of theinvention mean a membrane or membrane surface that adsorbs less thanabout 30 micrograms of IgG per square centimeter of membrane area asmeasured by the IgG binding test described herein.

As used herein in connection with the membranes of the presentinvention, the term “heat stable” as applied to the term “biomoleculeresistant surface” means a biomolecule resistant surface, for example amembrane surface, that, after exposure to heat as described herein, hasless than about twice the IgG adsorption of the same surface prior toheat exposure, as measured by the IgG test described herein.

In further preferred embodiments, the invention provides clean, causticresistant porous membranes comprising a porous substrate and aseparately formed heat stable biomolecule resistant surface. Preferably,the porous substrate is a membrane, more preferably a microporousmembrane.

As used herein in connection with the membranes and methods of thepresent invention, the term “clean membrane” means a membrane that, whenproduced, has either:

-   -   a. less than about 2 micrograms of extractable matter per square        centimeter of membrane, and preferably less than about 1        microgram of extractable matter per square centimeter, as        determined by the NVR Extraction test described herein; or    -   b. less than about 1 microgram of extractable matter per square        centimeter of membrane as determined by the TOC extractables        test described herein.

As used herein, the term “caustic resistant” as applied to membranes ofthe invention means a membrane that remains wettable after exposure to0.1 NaOH for two hours at ambient temperature, and has a ratio of flowtimes after such exposure to that before such exposure of less thanabout 1.5, when measured by the flow time measurement test describedherein.

A wide variety of porous media are useful in the practice of the presentinvention. Examples of such porous media include ceramics, metals,carbon and polymers. In some preferred embodiments, the porous medium isa polymer membrane. Representative polymers that can be used tomanufacture porous membranes useful in the present invention includepolysulfone polymers, preferably aromatic sulfone polymers, such aspolysulfone and polyethersulfone polymers, perfluorinated thermoplasticpolymers including polytetrafluoroethylene and polyvinylidenedifluoride, polyolefin polymers such as polyethylene, ultrahighmolecular weight polyethylene and polypropylene, and polyesters such aspolyethyleneterepthalate and polycarbonate. In some particularlypreferred embodiments, the porous membrane is a polyvinylidenedifluoride membrane. Those skilled in the art will readily be able toidentify other polymers useful in the formation of porous membranessuitable for the present invention.

In some preferred embodiments, the porous media or membrane ishydrophobic media or a hydrophobic membrane. In other preferredembodiments, the porous media or membrane is hydrophilic media or ahydrophilic membrane. In embodiments where the porous membrane ishydrophilic, polyamides, cellulose acetate and cellulose are preferred.

In some preferred embodiments, the heat stable biomolecule resistantsurface is formed on a porous membrane. As used herein, the term “porousmembrane” includes both microporous membranes and ultrafiltrationmembranes. The ultrafiltration and microporous membranes of theinvention can be in any of several forms, including sheets, tubes, andhollow fibers.

As used herein, the term “surface” as applied to the surface coatings ofthe membranes and methods of the invention shall mean the entire surfacearea of a porous media or membrane, including external surfaces and theinternal surface of the porous media or membrane. The term “externalsurface” means a surface that is exposed to view, for example either ofthe planar porous surfaces of sheet membranes. The term ‘internalsurface” is intended to denote the internal surface of a porous network,i.e., the interstitial area, of a porous media or membrane.

In general, porous membranes may be skinned or unskinned. A skin is arelatively thin, dense surface layer integral with the substructure ofthe membrane.

In skinned membranes, the major portion of resistance to flow throughthe membrane resides in the thin skin. In both microporous andultrafiltration membranes, the surface skin, where present, containspores leading from the external surface to the continuous porousstructure of the membrane below the skin. For skinned microporous andultrafiltration membranes, the pores represent a minor faction of theexternal surface area. In contrast, an unskinned membrane will be porousover the major portion of the external surface. The external surfaceporosity of the membrane (that is, the arrangement of pores of theexternal surface of the membrane as viewed by, for example, scanningelectron microscopy; “SEM”) can be single pores that are relativelyevenly distributed on the external surface of the membrane, or can bediscrete areas of porosity, or mixtures thereof. As used herein, theterm “surface porosity” as applied to an external surface of a membraneis the ratio of the area defined by the pore openings of the externalsurface to the total surface area of the external surface.

Microporous membranes useful in the practice of the present inventionmay be classified as symmetric or asymmetric, referring to theuniformity of the pore sizes across the thickness of the membrane, or,for a hollow fiber, across the porous wall of the fiber. As used herein,the term “symmetric membrane” indicates a membrane that hassubstantially uniform pore size across the membrane cross-section. Theterm “asymmetric membrane” is intended to mean a membrane in which theaverage pore size is not constant across the membrane cross-section. Forexample, in asymmetric membranes pore sizes can vary smoothly ordiscontinuously as a function of location through the membranecross-section. As will be appreciated, included within the definition of“asymmetric membranes” are membranes that have a ratio of pore sizes onone external surface to those on the opposite external surface that aresubstantially greater than one.

As used herein, the term “crosslinked terpolymer” means a polymer madefrom three or more monomers, of which at least one monomer has two ormore reactive sites which can take part in a polymerization reaction, orcan crosslink separate polymer chains. Terpolymers are generallyconsidered as being made from three monomers, but in the context of thepresent invention, terpolymers are not limited to three monomers, as itmay be desirable to use one or more additional monomers to impart orrefine desired properties of the membrane. In some preferredembodiments, the crosslinked terpolymer is made from two monofunctionalmonomers and one difunctional monomer.

The crosslinked terpolymer preferably covers the entire surface of theporous media or membrane. In preferred embodiments, the crosslinkedterpolymer is formed in situ from a solution of two or moremonofunctional monomers and a crosslinking polyfunctional monomer(referred to herein as the “reactant solution”). A monofunctionalmonomer is one that has a single unsaturated functional group.Polyfunctional monomers are molecules which have more than oneunsaturated functional group. Preferably, two or more of themonofunctional monomers are mono- or di-N-substituted acrylamides ormethacrylamides. The crosslinking monomer is preferably a polyfunctionalacrylamide or methacrylamide. In one particularly preferred embodiment,dimethylacrylamide and diacetone acrylamide are used withmethylene-bis-acrylamide. In another particularly preferred embodiment,N-vinyl pyrrolidone is substituted for one of the mono- ordi-N-substituted acrylamide or methacrylamide monofunctional monomers.

In some preferred embodiments, at least one polyfunctional monomer is apolyfunctional acrylamide monomer, a polyfunctional methacrylamidemonomer, or a diacroylpiperazine, and at least two differentmonofunctional monomers are selected from acrylamide monomers,methacrylamide monomers, and N-vinyl pyrrolidones.

In further preferred embodiments, at least one polyfunctional monomer isa polyfunctional acrylamide monomer, a polyfunctional methacrylamidemonomer, or a diacroylpiperazine, and at least two differentmonofunctional monomers are selected from N-vinyl pyrrolidone monomersand monomers having the general formula:

alternatively, described as H₂C═C(R¹)C(═O)N(R²) (R³)

-   -   wherein:    -   R¹ is —H or CH₃,    -   R² is H or C₁-C₆, preferably C₁-C₃ alkyl, either linear or        branched,    -   R³ is H or C₁-C₆, preferably C₁-C₃ alkyl, either linear or        branched, or C(CH₃)₂CH₂C(═O)CH₃, or (P═O)((NCH₃)₂)₂, or        C═ON(CH₃)₂, or CH₂—O—R⁴, where R⁴ is C₁-C₅ alkyl, either linear        or branched, or (CH₂—CH₂—O)n-R⁵, where R⁵ is —H or —CH₃, and n=2        or 3; provided that R² and R³ are not simultaneously H.

In some more preferred embodiments, the crosslinked terpolymer is apolymer formed from either:

-   -   (a) methylene-bis-acrylamide, dimethylacrylamide, and diacetone        acrylamide; or    -   (b) methylene-bis-acrylamide, -vinyl pyrrolidone, and either of        dimethylacrylamide or diacetone acrylamide.

Also provided in accordance with some preferred embodiments of thepresent invention are methods for the preparation of a clean, causticresistant porous membrane, said membrane comprising a porous membranesubstrate and a heat stable biomolecule resistant surface coating, saidmethod comprising the steps of

-   -   a. providing a porous membrane substrate;    -   b. optionally washing said porous membrane substrate with a        wetting liquid to wet the surfaces thereof;    -   c. optionally washing said wet porous membrane substrate with a        second wetting liquid to replace said first wetting liquid,        leaving said porous membrane substrate wetted with said second        liquid;    -   d. contacting the surfaces of said porous membrane substrate        with a solution containing:        -   (1) at least two monofunctional monomers selected from the            group consisting of acrylamides, methacrylamides, and            N-vinyl pyrrolidones; and        -   (2) at least one polyfunctional monomer selected from the            group consisting of polyfunctional acrylamides,            polyfunctional methacrylamides and diacroyl piperazines;        -   said solution optionally further comprising one or more            polymerization initiators;    -   e. polymerizing said monomers to form said heat stable        biomolecule resistant surface; and    -   f. washing said membrane.

In preferred embodiments, the entire surface of the porous media ormembrane is coated with the terpolymer. Thus, the reactant solutionshould preferably wet the entire surface of the porous media ormembrane. This is preferably facilitated by performing a washing stepprior to contacting the porous media or membrane with the reactantsolution. Thus, in some preferred embodiments, the porous media ormembrane is first washed with a washing liquid which completely wets theentire porous media or membrane surface. Preferably, the washing liquiddoes not swell or dissolve the porous media or membrane, and also canpreferably be exchanged with the reactant solution.

In some preferred embodiments where an aqueous reactant solution isemployed, the wetting liquid can be an organic-water composition havinglower surface tension than the surface tension required to wet theporous media or membrane. Examples of suitable wetting liquids arealcohol-water solutions, preferably methanol-water, ethanol-water, orisopropanol-water solutions.

In some preferred embodiments where a washing step is employed, it isdesirable to perform a second washing step. For example, where one ormore components of the wetting liquid can interfere with thepolymerization or crosslinking reactions, a second washing step can beused to remove the washing liquid and replace the same with a secondwashing liquid that does not interfere with the polymerization orcrosslinking reactions. For example, if an aqueous reactant solution isto be used, the wet porous media or substrate is washed with water toremove the first wetting liquid and produce a water filled porous mediaor membrane. The wet porous media or membrane is then contacted with thereactant solution (for example by soaking in the reactant solution) toproduce the desired reactant composition in the pores of the porousmedia or membrane, and on the external surfaces thereof. Preferably, thefirst and second washing steps, where desired, are performed at ambienttemperatures, for instance, 20° C. to 30° C., and preferably for timesfrom a few seconds to a few minutes.

If the reactant solution wets the porous media or membrane sufficiently,due to containing an organic solvent for that purpose, or if theconcentration of reactants in the reactant solution is sufficient tolower the surface tension of the solution to allow the reactant solutionto fully wet the porous media or membrane, then neither of the washingsteps are required. Thus, in some preferred embodiments, the reactantsolution will contain one or more additives which lower the surfacetension of the reactant solution sufficiently to avoid such washingsteps, and which do not interfere with the subsequent polymerizationreaction. Preferred examples of such additives include ethyl hexyl diol,propylene carbonate, tripropyleneglycol methyl ether and2-methyl-2,4-pentane diol. The amount of additive to the reactantsolution required to achieve proper wetting depends on the amount andtype of monomers and initiators being used, and will be readilydeterminable by those of skill in the art without undue experimentation.

In some preferred embodiments, the reactant solution includes solvent,mono functional monomers, at least one polyfunctional crosslinkingmonomer, and, optionally, one or more initiators. The choice of solventfor the reactant solution depends on the choice of monomers and optionalinitiators. The solvent preferably (1) dissolves the reactants and, ifpresent, the initiator; (2) does not interfere or hinder thepolymerization reaction; and (3) does not attack the porous media ormembrane. One example of a particularly preferred solvent is water.

In some especially preferred embodiments of the invention, theterpolymer is formed from at least two monofunctional monomers chosenfrom acrylamides, methacrylamides, or N-vinyl pyrrolidones, and at leastone polyfunctional acrylamide or methacrylamide crosslinking monomer.However, in other preferred embodiments, other monomers may be used.These include N-vinyl pyrrolidones, and other mono- or di-N-substitutedacrylamide or methacrylamide monomers, for example those having theformula:

alternatively, described as H₂C═C(R¹)C(═O)N(R²) (R³)

wherein:

R¹ is —H or CH₃,

R² is H or C₁-C₆, preferably C₁-C₃ alkyl, either linear or branched,

R³ is H or C₁-C₆, preferably C₁-C₃ alkyl, either linear or branched, orC(CH₃)₂CH₂C(═O)CH₃, or (P═O)((NCH₃)₂)₂ , or C═ON(CH₃)₂, or CH₂—O—R⁴,where R⁴ is C₁-C₅ alkyl, either linear or branched, or (CH₂—CH₂—O)n-R⁵,where R⁵ is —H or —CH₃, and n=2 or 3; provided that R² and R³ are notsimultaneously H.

In some embodiments, it is preferred that the ratio of a firstmonofunctional comonomer to a second monofunctional comonomer be fromabout 1 to 5, more preferably from about 1 to 2. In further preferredembodiments, the total amount of comomomers is from about 0.5% to about20%, more preferably between about 2% to about 10%, and still morepreferably from about 45 to about 8%.

In some preferred embodiments, the terpolymer can contain one or moremonofunctional monomers in addition to the two monofunctional monomerspreviously described. Such additional monofunctional monomers can beadvantageously employed to impart or modify specific properties of theterpolymer. For example, in some embodiments where it is desirable tomodify the hydrophilic nature or ionic charge content of the terpolymer,it is preferable to include a third monofunctional monomer having adifferent functionality from the other two monofunctional monomers toeffect the modification. Preferably, where an additional monofunctionalmonnomer or monomers are employed in the terpolymer, such additionalmonomers are employed in a minor amount, or an amount comparable to themonofunctional monomers. Representative additional property modifyingmonomers include can be(3-(methacryloylamino)propyl)-trimethylammoniumchloride, (3-acrylamidopropyl)trimethylammonium chloride,2-acrylamido-2-methyl-1-propanesulfonic acid andaminopropylmethacrylamide

In preferred embodiments if the invention, the terpolymer contains atleast one polyfunctional crosslinker monomer (or “crosslinkingmonomer”). While not wishing to be bound by a particular theory, it isbelieved that the crosslinking monomer facilitates a permanentmodification to the porous substrate or membrane by, inter alia, bothparticipating in the chain polymerization reactions, and by crosslinkingthe polymerized chains of monofunctional monomer. Examples of preferredcrosslinking monomers suitable for use in the present invention includepolyfunctional acrylamides, polyfunational methacrylamides, anddiacroylpiperazine, with polyfunctional acrylamides, and polyfunationalmethacrylamides being more preferred. Ethylene-bis-acrylamide andmethylene-bis-acrylamide are particularly preferred crosslinkingmonomers, with methylene-bis-acrylamide being especially preferred.

In some preferred embodiments, the ratio of amount of crosslinkermonomer to the total amount of monofunctional monomers present in theterpolymer is from about 1 to about 10, more preferably from about 2 toabout 6.

As used herein in reference to the monomeric components of theterpolymer, the terms “monomer” and “comonomer” shall be usedinterchangeably.

In some preferred embodiments, the polymerization of the monofunctionalmonomers and the crosslinking copolymer or copolymers of the presentinvention is achieved through free radical initiation and propagation.In some preferred embodiments, one or more free radical initiators canbe included in the in the reactant solution containing the monomers tofacilitate polymerization. Any of a wide variety of initiators known inthe art will find applicability in the present invention. In somepreferred embodiments the initiator or initiators are water soluble. Inother preferred embodiments, for example when wetting reactant solutionsare used, sparingly water soluble initiators are preferred. Those ofskill in the art will readily be able to determine suitable initiatorsfor a given reactant solution. Examples of suitable initiators include,for example, ammonium persulfate, potassium persulfate,azobis(4-cyanovaleric acid, Irgacure2959 (Ciba-geigy, Hawthorn, N.Y.),2,2′-azobis(2-amidino-propane)hydrochloride and the like. Preferably,the initiator or initiators are used in the range of from about 0.1% toabout 1% by weight, based on the total reactant solution.

In preferred embodiments, after the surface of the porous media ormembrane is contacted with (i.e., is saturated with) the reactantsolution, excess reactant solution removed from the external surfaces,while still leaving such external surfaces wetted with solution. Forsmall sheets, excess reactant solution can be removed by, for example,placing the saturated sheet between two layers of plastic film androlling out excess liquid with a rubber roll, such as. for example, ahand brayer. In processing continuous sheets of porous media ormembranes, removal of excess liquid can be performed with air knives,which direct a stream of air at the external surfaces. The force of theair stream sweeps away the excess reactant solution. One preferredtechnique is to run the sheet between two pressure controlled contactingrolls, at least one of which is elastomer coated, which rotate in thesame direction as the sheet. The amount of liquid left in the sheet canbe accurately controlled by adjusting the pressure of the contactingrolls.

After the excess reactant solution is removed, polymerization of thereactant solution is then begun by exposing the wet porous media ormembrane to any conventional energy source, such as heating, ultravioletlight, electron beam or gamma radiation. Free radical polymerizationinitiated by heat is typically achieved by heating the saturated mediaor membrane to at least about 60° C. and maintaining that temperaturefor from about 0.1 to about 10 minutes, preferably between about 1 toabout 2 minutes. Higher temperatures can be used depending on thecombination of initiator and monomers used, up to the point whereboiling or too rapid vaporization adversely affects the polymerizationreaction.

In some preferred embodiments, ultraviolet light is used to initiate thein situ polymerization reaction. Preferably, the porous media ormembrane saturated with the reactant solution (which optionally containsone or more initiators) is illuminated with an ultraviolet light sourcesuch as Fusion Systems F600 (Rockville, Md.) with an “H” bulb. Filterscan be used to reduce or remove undesirable wavelengths which may causeunwanted damage to the porous media or membrane being modified. Those ofskill in the art will appreciate that the balance of exposure time tothe UV lights with lamp intensity to optimize polymerization conditionswill be a matter of routine experimentation. Generally, with a 600 wattsource, exposure times of from about 2 seconds to about 10 seconds,preferably from about 3 seconds to about 5 seconds, will be suitable.

In some preferred embodiments, electron beam technology is used toinitiate polymerization, for example by methods described in U.S. Pat.No. 4,944,879, the disclosure of which is incorporated herein byreference. Typically, a web or individual sample is passed through acurtain of electrons generated by an electron beam processor. Theprocessor delivers the desired dose at from about 100 kV to about 200kV. The moving web or sample is transported at a speed suitable to givethe desired exposure time under the curtain. Exposure time, combinedwith dose, determines the dose rate. Typical exposure times are fromabout 0.5 seconds to about 10 seconds. Dose rates generally are from0.05 kGy (kiloGray) to about 5 kGy.

It is known that the presence of molecular oxygen adversely affects freeradical polymerization reactions. Thus, in each of the foregoing methodsof initiation previously described, it is preferred that the amount ofoxygen in the reaction zone be controlled to levels below about 200 ppm,preferably below about 50 ppm. In some preferred embodiments, this isaccomplished by flooding the reaction zone with inert gas such asnitrogen or argon, or by sandwiching the sheet between two layers ofplastic film to exclude air.

In further preferred embodiments, the polymerization of the monomers ofthe reactant solution can be initiated by gamma irradiation. Typically,in this method, a wound roll of monomer saturated porous membrane isirradiated. The roll can be passed through the reactant solution androlled up, or a previously wound up roll can be immersed in the reactantsolution. Preferably, the reactant solution is degassed, that is,treated so as to remove air, and particularly oxygen, from the solution.In some preferred embodiments, degassing is accomplished by replacingair with an inert gas such as helium, nitrogen or argon. In otherpreferred embodiments, degassing is accomplished by reducing thepressure over the monomer solution, for example with a vacuum pump. Thedegassed monomer solution laden roll is then sealed with a sealingmaterial so as to remain in a degassed state, and then irradiated at thedesired dose. Preferably, the sealing material will not be degraded bythe irradiation, and also does not significantly retard the gamma rays.A wide variety of materials are known in the art to be useful as sealingmaterials, for example many plastics, and borosilicate glass.

Typically, total dosages of about 0.02 to about 1.0 kGy are suitable.Typical exposures of about 5 to about 500 kilorads per hour, morepreferably about 5 to about 150 kilorads per hour can be used, withtypical irradiation times of from about 4 to about 60 hours. Those ofskill in the art will readily be able to determine the proper balance ofdose rate and time to arrive at the total dosage.

The degree to which the crosslinked terpolymer is grafted (i.e., thedegree to which the terpolymer is bound) to the porous media or membranecan be controlled by, inter alia, the choice of method of initiation thepolymerization reaction. For example, gamma irradiation gives a greaterdegree of grafting of the copolymer to a polymeric bulk matrix, whileheat induced initiation will have a lesser degree of grafting. Those ofskill in the art will readily be able to select the initiation methodthat will give the desired degree of grafting of terpolymer to theporous media or membrane.

The methods of the invention are applicable to the fabrication of sheet,tube and hollow fiber membranes. Coating methods are known from thetextile fiber and monofilament industries which can be adapted to thisprocess.

In some embodiments, the present invention provides membranes andarticles comprising polymer membranes, wherein the membranes include asurface coating which comprises at least onehydroxymethyldiacetoneacrylamide (HMDAA) monomer of formula:

wherein R₁ and R₂ are each independently H or CH₂OH. In some preferredembodiments, R₁ and R₂ are each CH₂OH. HMDAA is available from AldrichChem. Co., St Louis. Mo.

Membrane coatings incorporating HMDAA can be prepared by the methodsdescribed herein, as well as other methods known in the art for thepreparation of coated polymer membranes. For example, any of the manycrosslinnking monomers, for example methylenebisacrylamide (MBAm) can bepartially or completely replaced by HMDAA in coating regimes.

Typically, HMDAA is employed in sufficient amount to effect crosslinkingof the polymer coating. In some embodiments, the molar ratio of totalmonofunctional monomer in the surface coating to HMDAA is 10:1. In somemore preferred embodiments, the ratio is from 7:1 to 3:1. In somepreferred embodiments, the ratio is 4:1, 5:1, or 6:1.

In addition to the novel coated membranes described herein, any of avariety of polymer membranes known in the art can be coated with apolymer including HMDAA as a crosslinking monomer. Representativemembranes that can be modified by surface coatings incorporating theHMDAA monomers include all those described in this specification, andespecially, and without limitation, polysulfone polymers, preferablyaromatic sulfone polymers, such as polysulfone and polyethersulfonepolymers, perfluorinated thermoplastic polymers includingpolytetrafluoroethylene and polyvinylidene difluoride, polyolefinpolymers such as polyethylene, ultrahigh molecular weight polyethyleneand polypropylene, and polyesters such as polyethyleneterepthalate,polycarbonate, plasticized and nonplasticized polyvinyl chloride, andother thermoplastic polymers.

In some preferred embodiments, one or more HMDAA crosslinking monomersare completely or partially substituted for methylenebisacrylamide asthe crosslinking monomer in each of the novel crosslinked terploymersurface coated membranes described herein.

In some embodiments, HMDAA can be the sole crosslinking monomer of thesurface coating, and in other embodiments HMDAA is employed with one ormore additional crosslinking monomers.

While not wishing to be bound by any particular theory, it is believedthat when HMDAA (particularly wherein R₁ and R₂ are each CH₂OH) isheated, a vinyl ketone is generated at the end of the HMDAA moleculeopposite from the acrylamide portion thereof, providing a difunctionalacrylamide-vinyl ketone crosslinking monomer. HMDAA is further describedin, for example, the publication “HMDAA Monomer”, published by LubrizolChemical Co., incorporated herein by reference.

The HMDAA crosslinked surface coated membranes of the invention possessadvantages compared to other surface coated membranes prepared usingother crosslinking monomers. For example, it is believed that pendant—CH₂OH groups of the HMDAA monomers can persist after the crosslinkingreaction. These residual hydroxymethyl residues can advantageously beemployed as nucleophilic sites, which serve as post-coating formationpoints of attachment for incorporation of a variety of functionalmoieties into the coating. For example, such functional moieties can beattached to the coating via tethers having electrphilic moieties whichcan react with the HMDAA hydroxymethyl residues. Such electrophilicmoieties include, for example, glycidyl groups, α-ketomethylene groups,and isocyanates. Representative functional moieties which can beattached by the tethers include charged moieties such as quaternaryammonium species, sulfonic acids, and other charged groups useful forimparting charge to the membrane coating.

It is further believed that membranes employing surface coatingsincluding the HMDAA crosslinking monomers possess additional advantages.For example, the crosslinking monomer methylenebisacrylamide, whilesuitable for surface coatings as described herein, is believed in someinstances to result in increased protein binding and lower wettabilityrelative to other polymer constituents, and increased brittleness. Incontrast, the HMDAA crosslinking monomer is not believed to impart suchproperties.

It is not the intent of the discussion of the present invention toexhaustively present all combinations, substitutions or modificationsthat are possible, but to present representative methods for theedification of the skilled practitioner. Representative examples havebeen given to demonstrate reduction to practice and are not to be takenas limiting the scope of the present invention. The inventor seeks tocover the broadest aspects of the invention in the broadest manner knownat the time the claims were made.

Methods 1. Biomolecule Resistance

Biomolecule resistance to adsorption of porous membranes is measured bya static soak test using IgG protein. The protein solution is preparedwith phosphate buffered saline (PBS), purchased from Sigma ChemicalCompany, St. Louis. Mo. (Sigma I-5523). Goat gamma globulin, alsoobtained from Sigma Chemical Company (Sigma 1-5523) is used at aconcentration of 1 mg/ml. anti(rabbit IgG) is purchased from NEN LifeScience Products Boston, Mass. (NEX-155) and added to the proteinsolution to reach a final concentration of 0.1 μCi/ml.

One 13 mm membrane disk is placed in a 16×100 mm test tube and exactly 1ml of protein solution is added with a calibrated micropipettor. Alltest tubes are placed in a rack on a rotary shaker table and agitatedfor two hours. After agitation, the fluid is aspirated from the testtubes and the wet membrane is washed three times with 1 ml PBS. Thewashed membrane disk is transferred to a clean test tube and placed intoa gamma counter (Minaxi Auto-gamma 5000 series from Packard InstrumentCompany, Downers Grove, Ill.) to determine the radioactivity bound oneach disk, in units of counts per minute (cpm). Counts per minute forcontrol tubes with 1 ml of protein solution and no membrane are alsodetermined. Based on the control tubes, the relation between measuredradioactivity and actual protein concentration is calculated for theamount of protein on each disk by the following equation:

Control relation=total mean cpm/mg IgG=total mean cpm/1000 μg IgG

Because the radioactivity found on each disk is measured, the amount ofprotein on each disk can be calculated through the following equation:

Protein bound in one 13 mm disk=(cpm/1.33 cm²)*(1000 μg IgG/total meancpm)

This quantity is reported as protein bound in units of μg per cm² offrontal membrane area (a 13 mm disk has an area of 1.33 cm²). At leastduplicates are run for each sample tested.

Typical values found for low-binding commercial PVDF membranes, such asDurapore® membranes, are in the 15±4 μg/cm² range. In contrast,competitive PVDF membranes, such as Fluorodyne® membranes from Pallcorporation, are in the 47±19 μg/cm² range.

2. Heat Stability of Biomolecule Resistance

To determine the heat stability of the biomolecule resistance ofmembranes, samples of membranes were either (1) heated to 135° C. fortwo hours in an oven; or (2) held in a 121° C. steam autoclave for onehour, and then tested as described above.

3. TOC (Total Organic Carbon) Method for Determining Extractables Level

Three 47 mm disks of membrane are cut and placed in a pre-cleaned 40 mlTOC vial. The vial is covered by GVX (hydrophobic PVDF) and is securedby a rubber band. The vial is then autoclaved at 126° C. for 1 hour.After cooling the vial is removed. The GVX is removed and 40.0 ml offresh MilliQ® water is added, and the vial immediately capped with itspre-cleaned, Teflon lined septum cap. The membranes are allowed toextract overnight (minimum 16 hrs). The extracts are then analyzed forTOC levels by a Sievers 800 TOC analyzer. The raw PPM results arecorrected for the blank, which is an empty vial that was also autoclavedand extracted overnight. These ppm TOC results are converted to μg C percm² by multiplying by 40 ml and dividing by 52.05 cm².

4. TOC/NVR (Total Organic Carbon/Non-Volatile Residue) Method forDetermining Extractables Level for Production Scale Membranes

Approximately 8 ft² of membrane is coiled and then wrapped in GVX. Thesample is then autoclaved at 126° C. for 1 hour. After cooling themembrane is removed from the GVX and added to 800 ml of fresh MilliQ®water in a pre-cleaned 1 L graduated cylinder such that the membrane iscompletely submerged. The cylinder is capped with a layer of aluminumfoil and the membrane is allow to extract overnight (minimum 16 hours).The membrane is then removed. Aliquots of the extracts are then analyzedfor TOC (40 ml) and NVR (200-600 ml) by standard methods. The resultsare corrected for blanks and reported as μg C per cm2 and mg NVR per 7.5ft² respectively.

5. Flow Time Measurement to Determine Caustic Resistance

In this test modified membranes are tested for Flow Time, which is amethod to measure permeability, exposed to 0.1 NaOH for two hours atambient temperature and re-tested for Flow Time. The ratio of Flow Timesafter to before exposure is a measure of the effect of caustic on themembrane. A higher ratio shows more effect. A ratio of 1 shows noeffect. A caustic resistant membrane is one that remains wettable afterthis exposure and has a ratio of flow times after exposure to thatbefore exposure of less than about 1.5.

The following procedure is employed for this test:

1. Membranes are cut into 47mm disks.

2. The disks are wetted out with water and placed in a filter holderwith a reservoir for holding a volume of water and attached to a vacuumpump.

3. Water is flowed through the membrane under 27.5 inches Hgdifferential pressure.

4. After equilibrium was achieved, the time for 500 ml of water to flowthrough the membrane is recorded.

5. This measurement is made before and after exposure to 0.1M NaOH fortwo hours at ambient temperature. Exposure to 0.1M NaOH is performed byswirling the disks in a large excess of base, and washing the membraneswith water to neutrality before the flux measurement.

6. Data are rounded off the ratios to the nearest whole number.

EXAMPLE 1 Modification and Evaluation of PVDF Microporous Membranes byMethods of the Invention

Six 47 mm disks of the hydrophobic PVDF membrane of 0.1 micron rating(Durapore® Millipore Corporation, Bedford, Mass.) were cut and theirweight is recorded. They were then pre-wetted with water by being placedin methanol, and then soaked in MilliQ® water.

A solution was made containing the acrylamide monomers, photoinitiator,and water. The composition of this solution is shown in Table 1:

TABLE 1 Component Grams N,N-Dimethylacrylamide (DMAm) 1.50 gramsDiacetoneacrylamide (DACAm) 1.50 N,N′-Methylenebisacrylamide (MBAm) 0.75Irgacure 2979* photoinitiator 0.15 Water 96.1  100 total grams*Ciba-Geigy, Hawthorn, NY

After total dissolution of reactants, the solution is placed in a dishand the prewetted membranes are introduced into the solution. The dishis covered and the membranes are swirled on a Orbit shaker (LabLineInstruments, Melrose Park, Ill.) in the solution for 10 minutes. Themembranes are removed and individually placed between 1 mil polyethylenesheets. The excess solution is removed by rolling a rubber roller overthe polyethylene/membrane disks/polyethylene sandwich as it lays flat ona table. The polyethylene sandwich is then taped to a transport unitwhich conveys the assembly through a Fusion Systems UV exposure lab unitwith an “H” bulb. Time of exposure is controlled by how fast theassembly moves through the UV unit. In this example, the assembly movesthrough the UV chamber at 20 feet per minute.

After emerging from the UV unit, the membranes are removed from thesandwich and immediately placed in methanol, where they are washed byswirling for 15 minutes. Next, they are washed in MilliQ® water for 15minutes. Following this washing procedure they are allowed to air dry.

The membranes are weighed again, and the % weight add-on per membrane iscalculated as the added weight divided by the initial weight multipliedby 100. For this example, the weight add-on was 4.4%.

Table 2 shows the measurements made on the membranes:

TABLE 2 Membrane Number Conditioning Measurements One None Wetting time,flow time, protein binding Two Dry heat Wetting time, protein for 2hours at 135° C. binding Three Autoclave Wetting time, protein 1 hour at124° C. binding Four, Five, Six None Total organic carbon extractables

Relative to the phobic substrate, the modified membrane displayed awater flow time increase of 25%. The total organic carbon extractableswere measured as 0.646 micrograms/square centimeter. The wetting timeand protein binding are given in Table 3 below:

TABLE 3 Membrane Number Wetting Time (seconds) Protein Binding (μg/cm²)One 0.2 14 Two 2.0 18 Three 0.3 21

Wetting time was measured by dropping a 47 mm disk horizontally intowater and timing the appearance of complete wetting through the disk.

EXAMPLE 2

Modification of PVDF Membranes Using Different Monomers with DMAm andMBAm

PVDF membranes were modified as described in Example 1 using differentmonomers with DMAm and MBAm, as shown Table 4 below:

TABLE 4 Protein Protein Binding Binding after Mono- Mono- Mono- Proteinafter Auto- Ex. mer/ mer/ mer/ Binding Dry Heat clave # grams gramsgrams as made μ/cm² μ/cm² TOC 2-1 DMAm/ DACAm/ MBAm/ 15 20 26 0.518 2.01.0 0.75 2-2 DMAm/ DEAm/ MBAm/ 13 34 20 2.0 1.0 0.5 2-3 DMAm/ IPAm/MBAm/ 16 30 21 2.0 1.0 0.5 2-4 DMAm/ BACAm/ MBAm/ 14 18 10 2.0 1.0 0.52-5 NVP/ DACAm/ MBAm/ 26 219 85 1.5 1.5 1.0 2-6 DMAm/ MBAm/ 19 199 720.661 3.0 1.0 2-7 NVP/ MBAm/ 15 305 217 1.5 1.0

EXAMPLE 3 Continuous Roll Production

Rolls of surface modified membrane were prepared by sequentially passinghydrophobic 0.1 μm pore sized membrane at various speeds through awetting pan containing an aqueous solution composed of variousconcentrations of N,N-dimethylacrylamide (DMAm),N,N-methylenebisacrylamide (MBAm), diacetoneacrylamide (DACAm), andtri(propyleneglycol)methyl ether (TPM, constant 20%), exposing to UVradiation from both sides using four Fusion UV Systems F600 lamps whilesandwiched between polyethylene film, rinsing in a tank of methanolfollowed by a tank of water, and drying over a vacuum drum whileimpinging with 115° C. dry air. The various conditions employed andresults are given in Table 5 below. Controls in Lines 1 and 2 areincluded to demonstrate the effect of mixing monomers on the thermalstability of protein binding.

TABLE 5 Wettability dynes/cm to IgG Binding IgG Binding Water wet after2 hours IgG after 2 hrs @ after 131° C. % % % Speed Permeability 135° C.dry Binding 135° C. autoclave DMAm MBAm DACAm mm/sec LMH/kPa ovenexposure μg/cm² μg/cm² μg/cm² 3.0 1.0 0.0 102 21.0 82 27.0 139.9 100.30.0 1.0 3.0 76 28.7 127.4 29.2 0.75 0.75 4.0 102 54.5 60 19.1 27.2 19.90.75 0.75 4.0 102 51.4 60 16.9 27.2 19.3 0.75 0.75 4.0 152 52.1 70 14.326.1 13.6 0.75 0.75 4.0 152 53.0 70 16.0 27.7 19.6 1.00 1.00 5.0 15248.3 70 10.8 20.2 16.9 2.00 0.75 4.0 152 48.1 77 13.0 17.2 14.5 2.000.75 3.0 152 52.2 77 8.9 18.8 16.4 2.00 1.00 4.0 152 47.9 77 9.3 16.814.9

EXAMPLE 4

Results for Caustic Resistance Testing with Commercial PVDF Membranesand Those of the Present Invention

The ratio of Flow Times for membranes before and after exposure to 0.1 NNaOH for two hours at ambient temperature are shown in Table 6 below.

TABLE 6 Flow time after Membrane Flow Time (sec) exposure (sec) RatioDurapore ® 238 970 4 Fluorodyne ® 0.1μ 746 8491 11 Fluorodyne ® 0.2μ 2012987 15 Present Invention 281 301 ~1

As discussed above, it is greatly preferred that modified membrane, suchas those of the present invention, can be easily and effectively“cleaned”; i.e., subjected to a regime of post reaction washing and/orextraction steps to remove materials which could subsequently beextracted into the product being filtered. To reduce extractables tosuch low levels, it is necessary to have a cleaning method that willremove low molecular weight species and unreacted monomers, as well asunbound oligomers and polymeric species. This may be done bysufficiently exhaustive extraction with a liquid which will solvatethese materials. There is, however, an economic price to be paid as theextraction time is increased, and it is therefore desirable that theextraction time be minimized. Moreover, it has been observed thatmodified membranes of the prior art do not show reduced extractablesdespite lengthy washing or soaking, if they are subsequently autoclaved.While not wishing to be bound by a particular theory, it is believedthat this may be due to hydrolytic breakdown products of themodification resulting from autoclaving, which are extractedsubsequently. It can be seen that it would be desirable to have amodified membrane or porous media that had a stable extractables levelafter exposure to heat, for example autoclaving, or exposure to highlyalkaline liquids. It is a significant attribute of the membranes of thepresent invention that they can be efficiently cleaned up to very lowlevels of extractables, levels lower than membranes of the prior art.

FIG. 1 illustrates the results from a test to compare extractable levelsof membranes of the prior art and those of the present invention. Setsof both membranes were subjected to a post-manufacturing soak atconditions designed to remove significant amounts of any extractablespresent. Sub-sets of each type were soaked for different times in orderto determine the effect of increasing soak time on extractables removal.

Samples of commercial PVDF microporous membrane (Durapore®, MilliporeCorporation, Bedford, Mass.) were soaked in 80° C. water for times asshown in FIG. 1. After each time interval, samples were removed andtested in the TOC method for determining extractables levels. FIG. 1shows that TOC extractables are between about 2 to 5 micrograms persquare centimeter. The level of extractables was unaffected by the 80°C. soak time. This indicates a limit to any improvement for thesemembranes.

Membranes of the present invention were soaked in methanol for times asshown in FIG. 1, and TOC extractables were determined as describedabove. The black squares denote Durapore® and the grey triangles,inverted triangles and circles denote nidependent runs of identicalmembranes of the invention. It can be seen that for the membranes of theinvention, TOC extractables are approximately an order on magnitudelower, about 0.2 to 0.4 micrograms per square centimeter. These resultsshow that membranes of the present invention can be made significantlycleaner than those of the prior art, and that exhaustive soaking willnot lower the extractables level of membranes of the prior art to thelevel attainable for those of the present invention.

As discussed above, one significant problem with the membranes of theprior art is that their biomolecule resistance will significantlydecrease when heated; i.e., that is, their biomolecule resistance willnot be sufficiently heat stable. Dry heat has a more deleterious effect,and wet heat, as in autoclaving, has a lesser, but still substantialeffect. Since membranes are subjected to various heat regimes, dependingon use, these effects pose a serious drawback to commercial development.

Surprisingly, it has been discovered that the crosslinked terpolymers ofthe invention, and in particular the crosslinked polymers ofacrylamides, do not have this sensitivity to heat. In particular,crosslinked terpolymer containing methacrylamides and containing N-vinylpyrrolidones share this heat stability of high resistance to biomoleculesorption.

FIG. 1, which illustrates the results of Example 1, clearly shows thebeneficial effect of the present invention. In FIG. 2, the results fromthree modifications of the same base membrane are shown. Modification oftwo sets of samples with crosslinked polyacrylamide from a singlemonomer (N, N-dimethylacrylamide (DMAm) in one case and diacetoneacrylamide (DACAM) in the other) and crosslinker monomer (methylenebis-acrylamide (MBAm)) have good, that is low, protein adsorption asmade. When samples of these modified membranes are heated to 135° C. fortwo hours in an oven and then tested, protein adsorption was markedlyhigher, by approximately 4-10 times the as-made value. A separate sampleof each was held in a 121° C. steam autoclave for one hour and thentested for protein adsorption. The DMAm sample was much higher than theas-made value, while the DACAm sample had about the same value. In sharpcontrast, the membrane modified with a mixture of DMAm and DACAmmonomers showed a negligible effect from the two hour 135° C. heating orthe autoclaving. In addition, the as-made protein adsorption of theterpolymer modified membrane was lower than those of the single monomermodified membranes. A heat stable biomolecule resistant surface is onethat has no increase or a small increase in biomolecule adsorption afterheat exposure. More specifically, a heat stable biomolecule resistantsurface is one which, after exposure to heat as described herein, hasless than about twice the IgG adsorption of the same surface prior toheat exposure, as measured by the IgG test described herein.

A decrease in biomolecule adsorption after heat exposure may be seen dueto variability in the test method when the change is inherently small.There also may be optimized formulations that will give the effect of adecrease. In the context of the present invention, biomoleculeresistance is measured with the IgG test. Representative heat exposuresare done with dry heat at 135° C. for 2 hours, and with wet heat at 124°C. for 1 hour. A heat stable biomolecule resistant surface is one thathas less than about twice the IgG adsorption of the unexposed-to-heatsurface.

Given that practitioners in the field generally attempt to maximize thehydrophilic nature of surfaces where resistance to biomoleculeadsorption is desired, it is further surprising that in the Examples 2-1to 2-7 the use of a less hydrophilic monomer with the hydrophilicdimethylacrylamide monomer gives an improvement in overall biomoleculeresistance. Table 7 below gives the values of P, the octanol-waterpartition coefficient for representative monomers used in thisinvention. P equals the ratio of the concentration in the octanol phaseto the concentration in the aqueous phase when a compound is subjectedto an octanol-water two phase extraction. Higher values of P show a morehydrophobic compound.

TABLE 7 Monomer Abbreviation Monomer name P DMAm Dimethylacrylamide 4.57NVP -Methyl Pyrrolidine 3.80 MBAm N,N-Methylene bis- 2.63 acrylamideDACAm Diacetoneacrylamide 11.48 DEAM Diethylacrylamide 22.39 IPAmN-Isopropylacylamide 11.48 BACAm N-t-Butylacrylamide 13.49

P values for the monomers were calculated using ChemPlus® software(HyperCube, Inc., Waterloo, Ont.). Monomer P values were then used tocalculate a combined P value for the crosslinked terpolymers of Examples1 and 3. The combined values were the sum of the mass fraction of themonomers times its P value. The combined P value is a measure of thehydrophilic nature of the resulting polymer, the higher the value, theless hydrophilic is the polymer.

Table 8 shows the combined P values, the protein binding after dry heat(2 hours at 135° C.) and after autoclave (1 Hour at 124° C. steam)exposure. In all the examples in this Table, N,N-methylenebis-acrylamide is the crosslinking monomer.

TABLE 8 Protein Binding Protein Binding after Dry Heat after AutoclaveEx. # μ/cm² μ/cm² Combined Octanol- Water Partition Coefficient forCrosslinked Terpolymers DMAm/DACAm 6.06 20 26 DMAm/DACAm 6.96 18 21DMAm/DEAm 9.48 34 20 DMAm/IPAm 6.32 30 21 DMAm/BACAm 6.90 18 10DACAm/NVP 6.38 219 85 Combined Octanol- Water Partition Coefficient forCrosslinked Single Monomer Polymers DMAm 4.1  199 72 NVP 3.5  305 217

These examples are not shown to limit this invention, but to illustratethe surprising observation that the heat stability of resistance tobiomolecule adsorption, as here shown by protein binding after eitherdry or wet heat exposure, is improved by the incorporation of a morehydrophobic monomer into the crosslinked terpolymer. Specifically, theresults show that polymers made with MBAm and DMAm bind more proteinthan crosslinked terpolymers made with these monomers and a morehydrophilic monomer. Similarly, a crossinked polymer from NVP and MBAmhas more binding after heat exposure than a crosslinked terpolymer madewith NVP, MBAm and DACAm.

EXAMPLE 5 COMPARATIVE EXAMPLE 1 Sartorius Hydrosart 0.2μ CelluloseMembrane

Hydrosart 0.2μ cellulose membrane was removed from a Sartocon stack. Theethanol was washed out with MilliQ® water. After 3 washes with excessMilliQ® water, a sample of membrane was dried and analyzed by infrared.IR and Scanning Electron Microscopy indicated the membrane was composedof regenerated cellulose on a support of cellulose paper.

Alkaline Stability

The washed, but not dried membrane was subjected to the alkalinestability test. The ratio of flow times after to before base treatmentwas 1.06. This indicates that, according to this criterion, the membraneis alkaline stable.

Protein Binding

The membrane was tested for protein binding using the standard IgGprotocol. The values were:

-   As is: 183 μgrams/cm²-   2 hours 135° C. dry heat: 191 μgrams/cm²-   Autoclaved: 204 μgrams/cm²

Total Organic Carbon (TOC)

47 mm disks were washed for 30 minutes with MilliQ® water. The MilliQ®water was replaced, and the membranes were washed an additional 24hours. Through each individual disk a total of 275 cc of MilliQ® waterwere flushed under a pressure gradient of about 13 pounds/in². Thesemembranes were tested for TOC. Three membranes were grouped together andtested for TOC without autoclaving, and three membranes were groupedtogether and tested for TOC after autoclaving (the membranes of thepresent invention are tested for TOC after autoclaving). The experimentwas repeated to confirm the results (Table 9):

TABLE 9 TOC before TOC after Autoclaving autoclaving Membrane μgrams/cm²μgrams/cm² Hydrosart 0.2μ MF 0.25 5/08 TOC#1 Hydrosart 0.2μ MF 0.09 3.46TOC#2

This example indicates that although Hydrosart membranes can be flushedto a low TOC value, autoclaving generates large amounts of extractables.The membranes of the current invention do not generate extractables ofthis magnitude after autoclaving.

EXAMPLE 6 COMPARATIVE EXAMPLE #2 Sartorious Sartobran 0.2μ CelluloseAcetate Membranes

Sartobran P 0.2μ cellulose membrane was removed from a Sartocon stack.The membrane was washed with MilliQ® water. After 3 washes with excessMilliQ® water, a sample of membrane was dried and analyzed by infrared.IR and Scanning Electron Microscopy indicated the membrane was composedof cellulose acetate on a support of polyethyleneterphthalate.

Alkaline Stability

The washed, but not driec membrane was subjected to the alkalinestability test. The ratio of flow times after to before base treatmentwas 8.20. This indicates that, according to this criterion, the membraneis not stable to alkaline treatment.

Protein Binding

The membrane was tested for protein binding using the standard IgGprotocol. The values were:

-   A is: 62 μgrams/cm²-   2 hours 135° C. dry heat; 64 μgrams/cm²-   Autoclaved: 100 μgrams/cm²

Total Organic Carbon (TOC)

47 mm disks were washed for 30 minutes with MilliQ® water. The MilliQ®water was replaced, and the membranes were washed an additional 24hours. The membranes were tested for TOC using the standard autoclavemethod. This test gave a value of 2.35 μgrams/cm².

While the present invention has been illustrated in terms of porousmembranes, the methods of the invention are applicable to thepreparation of a variety of articles having the caustic resistant, heatstable biomolecule resistant surface described herein. Thus, in somepreferred embodiments, the present invention provides methods forpreparing an article having the caustic resistant, heat stablebiomolecule resistant terpolymer surface described herein. In someembodiments, the methods comprise contacting the surface of the articlewith a reactant solution containing:

(1) at least two monofunctional monomers selected from the groupconsisting of acrylamides, methacrylamides, and N-vinyl pyrrolidones;and

(2) at least one polyfunctional monomer selected from the groupconsisting of polyfunctional acrylamides, polyfunctional methacrylamidesand diacroylpiperazines;

said solution optionally further comprising one or more polymerizationinitiators;

polymerizing said monomers to form the heat stable biomolecule resistantsurface on the surface of the article, and optionally washing thearticle.

Those of skill in the art will appreciate that the invention will havewide applicability in, for example, the biotechnology industry, forexample in equipment that comes into contact with biological solutions.

HMDAA EXAMPLES

A mixed acrylamide formulation used to surface modify a 0.2μ hydrophobicPVDF Durapore membrane was altered by stepwise replacement of componentswith HMDAA (R₁ and R₂═CH₂OH). The formulations are shown below in Table10:

TABLE 10 Formulation DMAm DACAm MBAm HMDAA Control 2.25 2.25 0.75 0 12.25 2.025 0.75 0.225 2 2.25 1.125 0.75 1.125 3 2.25 0 0.75 2.25 4 2.252.25 0.675 0.075 5 2.25 2.25 0.375 0.375 6 2.25 2.25 0 0.75

Formulations 1-3 show replacement of DACAm at 10, 50 and 100%, whileformulations 4-6 show the replacement of MBAm at 10, 50 and 100%.Wettability and protein binding were tested (a) as formed; (b) dryheated at 135° C., and (c) autoclaved.

Relative to control, all test membranes better wetting after dry heat.The dry heated control wet slowly over 2 minutes, while the test sampleswetted at times from 1 to 10 seconds. Thus, even a 10% replacement ofeither DACAm or MBAm resulted in enahnced dry wettability.

Formulation 6 contains no MBAm, but still showed a weight percent add-onof more than 4%, thus confirming HMDAA as a crosslinking agent thatimparts enhanced wettability.

Protein binding experiments showed that the “as formed” protein bindingof all membranes were low. Removing DACAm from the formulation resultsin high dry heat protein binding. When DACAm is present at 2.25%,however, all membranes show low binding after dry heat. The results areshown in FIGS. 3 and 4.

The reference works, patents, patent applications, and scientificliterature, and other printed publications that are mentioned ofreferred to herein are hereby incorporated by reference in theirentirety.

As those skilled in the art will appreciate, numerous changes andmodifications may be made to the preferred embodiments of the inventionwithout departing from the spirit of the invention. It is intended thatall such variations fall within the scope of the invention.

1-66. (canceled)
 67. A method for the preparation of a clean, causticresistant porous membrane, said membrane comprising a porous membranesubstrate and a heat stable biomolecule resistant surface coating, saidmethod comprising the steps of: a. providing a porous membranesubstrate; b. optionally washing said porous membrane substrate with awetting liquid to wet the surfaces thereof; c. optionally washing saidwet porous membrane substrate with a second wetting liquid to replacesaid first wetting liquid, leaving said porous membrane substrate wettedwith said second liquid; d. contacting the surfaces of said porousmembrane substrate with a solution containing polyfunctional monomerthat is methylene-bis-acrylamide, and at least two monofunctionalmonomers, wherein at least one of said monofunctional monomers isdimethylacrylamide or diacetone acrylamide; and e. polymerizing saidmonomers to form said heat stable biomolecular resistant surface. 68.The method of claim 67 wherein said porous membrane substrate is apolyvinylidene difluoride microporous membrane.
 69. The method of claim67 wherein the solution further comprises a supplemental propertymodifying monomer.
 70. The method of claim 69 wherein said supplementalproperty modifying monomer is selected from the group consisting of(3-(methacryloylamino)propyl)trimethylammonium chloride,(3-acrylamidopropyl)trimethylammonium chloride,2-acrylamido-2-methyl-1-propanesulfonic acid andaminopropylmethacrylamide.
 71. The method of claim 69 wherein two ofsaid monofunctional monomers are present in the ratio of about 1 toabout 5 by weight.
 72. The method of claim 69 wherein two of saidmonofunctional monomers are present in the ratio of about 1 to about 2by weight.
 73. The method of claim 69 wherein the total amount of saidmonofunctional monomers present is from about 0.5% to about 20% byweight.
 74. The method of claim 69 wherein the total amount of saidmonofunctional monomers present is from about 2% to about 10% by weight.75. The method of claim 69 wherein the total amount of saidmonofunctional monomers present is from about 4% to about 8% by weight.76. The method of claim 69 wherein the ratio of the total amount ofmonofunctional monomers to polyfunctional monomer is about 2 to about 10by weight.
 77. The method of claim 69 wherein the ratio of the totalamount of monofunctional monomers to polyfunctional monomer is about 2to about 6 by weight.
 78. The method of claim 69 wherein the heat stablebiomolecule resistant surface is hydrophilic.
 79. The membrane of claim69, wherein said membrane has a biomolecule binding of less than about30 microgram per square centimeter measured by the IgG binding test. 80.The membrane of claim 69, wherein the membrane has TOC extractables ofless than about 1 microgram of extractable matter per square centimeterof membrane as measured by the TOC Extractables test.
 81. The membraneof claim 69, wherein the membrane has TOC extractables of less thanabout 2 micrograms of extractable matter per square centimeter ofmembrane as measured by the NVR Extractables test.
 82. The membrane ofclaim 69, wherein the membrane has caustic resistance of less than about1.3 as measured by the Flow Time Measurement test.
 83. The method ofclaim 69 wherein the sizes of the pores of the porous membrane substrateprior to performing steps (a) through (e) are substantially equivalentto the sizes of said pores after performing steps (a) through (e). 84.The method of claim 69 wherein said porous membrane substrate is amicroporous membrane.
 85. The method of claim 84 wherein saidmicroporous membrane is formed from one or more of the group consistingof aromatic sulfone polymers, polytetrafluoroethylene, perfluorinatedthermoplastic polymers, polyolefin polymers, ultrahigh molecular weightpolyethylene, and polyvinylidene difluoride.
 86. The method of claim 69,wherein one of said at least two monofunctional monomers isdimethylacrylamide.
 87. The method of claim 69, wherein one of said atleast two monofunctional monomers is diacetone acrylamide.